Wedlock D N, Aldwell F E, Buddle B M
AgResearch, Wallaceville Animal Research Centre, Upper Hutt, New Zealand.
Immunol Cell Biol. 1996 Apr;74(2):151-8. doi: 10.1038/icb.1996.20.
Immune responses in the Australian common brushtail possum (Trichosurus vulpecula) and in particular the role of cytokines are poorly understood. We have undertaken to isolate cytokine genes using reverse transcriptase-polymerase chain reaction (RT-PCR) and in this study describe the molecular cloning of TNF-alpha. Primers were designed from consensus sequences at the N-terminus end of eutherian mammalian TNF-alpha and the possum cDNA, derived from spleen RNA, identified by RT-PCR. The complete cDNA encoding possum TNF-alpha was amplified from lymphocyte RNA by 5' and 3' rapid amplification of cDNA ends (RACE). The nucleotide sequence of the protein coding region of this cDNA shared 66-69% identity with other mammalian TNF-alpha genes. The predicted protein of 233 amino acids shared 56-58% identity with eutherian mammalian TNF-alpha was expressed in both Saccharomyces cerevisiae and Escherichia coli by constructing expression plasmid derivatives of the vectors pYES2 and pGEX-2T respectively. Cell extracts prepared from transformants and the purified GST/TNF-alpha fusion protein exhibited cytotoxic activity on the TNF-alpha-sensitive murine fibroblast L929 cells and stimulated proliferation of possum thymocyte cells. The induction of possum TNF-alpha mRNA in alveolar macrophages was analysed by RT-PCR using possum-specific TNF-alpha primers. Macrophages cultured in the presence of LPS showed enhanced transcription of TNF-alpha mRNA. This is the first report of the cloning and sequence analysis of the cDNA encoding a marsupial cytokine gene.
澳大利亚帚尾袋貂（Trichosurus vulpecula）的免疫反应，尤其是细胞因子的作用，目前了解甚少。我们已着手使用逆转录聚合酶链反应（RT-PCR）分离细胞因子基因，并在本研究中描述肿瘤坏死因子-α（TNF-α）的分子克隆。根据真兽亚纲哺乳动物TNF-α N端的共有序列设计引物，通过RT-PCR从袋貂脾脏RNA中鉴定出袋貂cDNA。通过5'和3' cDNA末端快速扩增（RACE）从淋巴细胞RNA中扩增出编码袋貂TNF-α的完整cDNA。该cDNA编码区的核苷酸序列与其他哺乳动物TNF-α基因的同源性为66%-69%。通过分别构建载体pYES2和pGEX-2T的表达质粒衍生物，在酿酒酵母和大肠杆菌中表达了预测的233个氨基酸的蛋白质，该蛋白质与真兽亚纲哺乳动物TNF-α的同源性为56%-58%。从转化体中制备的细胞提取物和纯化的GST/TNF-α融合蛋白对TNF-α敏感的小鼠成纤维细胞L929具有细胞毒活性，并刺激袋貂胸腺细胞增殖。使用袋貂特异性TNF-α引物通过RT-PCR分析肺泡巨噬细胞中袋貂TNF-α mRNA的诱导情况。在LPS存在下培养的巨噬细胞显示TNF-α mRNA转录增强。这是关于有袋类动物细胞因子基因cDNA克隆和序列分析的首次报道。
